JP7681288B2 - Two-component curing adhesive, curing agent for two-component curing adhesive, and compound - Google Patents

Description

The present invention relates to a two-component curing adhesive, a curing agent for a two-component curing adhesive, and a compound.

Genipin has a lower cytotoxicity than glutaraldehyde and is used as a hardener for medical gels, including xerogel. Patent Document 1 describes a "sponge for hemostatic use, comprising genipin and gelatin."

JP 2018-68723 A

Genipin is difficult to use as a curing agent for two-component curing adhesives because the crosslinking reaction proceeds slowly. Therefore, the present invention aims to provide a two-component curing adhesive that contains a genipin derivative and can be cured quickly.
Another object of the present invention is to provide a curing agent and a compound for a two-component curing adhesive.

As a result of extensive research into achieving the above object, the inventors have discovered that the above object can be achieved by the following configuration.

[1] A two-component curing adhesive comprising a first agent containing a compound A having, in the molecule, at least one primary amino group and at least one group represented by formula 1, and a basic compound, and a second agent containing an acidic compound.
[2] The two-component curing adhesive according to [1], wherein at least one selected from the group consisting of the first agent and the second agent contains a solvent, and when the first agent and the second agent are mixed, the pH of the mixture is 8.0 or less.
[3] The two-component curing adhesive according to [2], wherein the pH of the mixture is less than 7.0.
[4] The two-component curing adhesive according to [2] or [3], wherein the pH of the mixture is 5.0 or higher.
[5] The two-component curing adhesive according to any one of [1] to [4], wherein the second component contains a solvent and has a pH of less than 7.0.
[6] The two-component curing adhesive according to [5], wherein the second component has a pH of 2.0 to 5.0.
[7] The two-component curing adhesive according to any one of [1] to [6], wherein the first component contains a solvent and has a pH of greater than 7.0.
[8] The two-component curing adhesive according to any one of [1] to [7], wherein compound A has a skeletal structure, the primary amino group bonded to the skeletal structure, and a group represented by formula 1 described below, and the skeletal structure is a skeletal structure derived from at least one compound selected from the group consisting of polysaccharides, peptides, proteins, gelatin, ethylenediamine, polyethyleneimine, polyalkylene glycols, polylactic acids, polyglycolic acids, polycaprolactones, and polyacrylic acids.
[9] The two-component curing adhesive according to any one of [1] to [8], wherein compound A is a compound represented by formula 2 described below.
[10] The two-component curing adhesive according to [9], wherein L ¹ , L ² , and L ³ each contain an alkyleneoxy group having 1 to 10 carbon atoms.
[11] The two-component curing adhesive according to any one of [1] to [10], wherein compound A is a compound represented by the following formula 5:
[12] The two-component curing adhesive according to any one of [1] to [11], wherein the second component contains an extracellular matrix.
[13] The two-component curing adhesive according to any one of [2] to [7], wherein the pH of the first and second components when mixed together is 5.0 to 8.0.
[14] The two-component curing adhesive according to any one of [1] to [13], wherein the ratio of the molar content of the group represented by the above formula 1 to the molar content of the primary amino group in the compound is 0.05 to 1.2.
[15] The two-component curing adhesive according to any one of [1] to [14], wherein compound A has 2 to 6 primary amino groups and 2 to 6 groups represented by formula 1.
[16] A curing agent for a two-component curing adhesive, comprising compound A having, in the molecule, at least one primary amino group and at least one group represented by formula 1.
[17] The curing agent according to [16], further comprising a basic compound.
[18] The curing agent according to [16] or [17], wherein the compound A has a skeletal structure, the primary amino group bonded to the skeletal structure, and a group represented by the formula 1, and the skeletal structure is derived from at least one compound selected from the group consisting of polysaccharides, peptides, proteins, gelatin, ethylenediamine, polyethyleneimine, polyalkylene glycols, polylactic acids, polyglycolic acids, polycaprolactones, and polyacrylic acids.
[19] A compound represented by formula 2 described below.

According to the present invention, it is possible to provide a two-component curing adhesive that contains a genipin derivative and can be cured rapidly.
The present invention also provides a curing agent and compound for a two-component curing adhesive.

FIG. 13 is a graph showing the results of calculating the amount of genipin introduced (vertical axis: pieces) versus reaction time (horizontal axis: minutes). FIG. 1 is a graph showing the results of calculating the average amount of genipin introduced per one molecule of polyamine versus the pH of the polyamine solution. 1 is a graph showing the change in shear modulus (Pa) over time, with the time when the first and second parts of the adhesive are mixed being set at 0 minutes, where solid circles indicate the storage modulus and hollow circles indicate the loss modulus. FIG. 13 is a graph showing the change in shear modulus (Pa) over time for only the second agent. FIG. 1 is a graph showing the change in shear modulus (Pa) over time, with the mixing time of the second agent and genipin set at 0 minutes. 6 is a graph showing the change in shear modulus (Pa) over time, with the mixing time of the second agent, genipin, and polyamine set at 0 minutes. The solid circle indicates the storage modulus, and the hollow circle indicates the loss modulus. The graph shows the change in shear modulus (Pa) over time when the first and second parts of the adhesive used in this adhesive were mixed at 0 minutes, using compound A synthesized by adding 50 mol% genipin to 100 mol% of the primary amino group of polyamine. The solid circle indicates the storage modulus, and the hollow circle indicates the loss modulus. The graph shows the change in shear modulus (Pa) over time when the first and second parts of the adhesive used in this adhesive were mixed at 0 minutes, using compound A synthesized by adding 60 mol% genipin to 100 mol% of the primary amino groups of the polyamine. The solid circle indicates the storage modulus, and the hollow circle indicates the loss modulus. The graph shows the change in shear modulus (Pa) over time when the first and second parts of the adhesive used in this adhesive were mixed at 0 minutes, using compound A synthesized by adding 70 mol% genipin to 100 mol% of the primary amino group of the polyamine. The solid circle indicates the storage modulus, and the hollow circle indicates the loss modulus. The graph shows the change in shear modulus (Pa) over time when the first and second parts of the adhesive used in this adhesive were mixed at 0 minutes, using compound A synthesized by adding 80 mol% genipin to 100 mol% of the primary amino groups of the polyamine. The solid circle indicates the storage modulus, and the hollow circle indicates the loss modulus. The graph shows the change in shear modulus (Pa) over time when the first and second parts of the adhesive using compound A obtained when the reaction time between genipin and polyamine was 3 minutes was set to 0 minutes. The solid circle indicates the storage modulus, and the hollow circle indicates the loss modulus. The graph shows the change in shear modulus (Pa) over time when the first and second parts of the adhesive using compound A obtained when the reaction time between genipin and polyamine was 10 minutes was set to 0 minutes. The solid circle indicates the storage modulus, and the hollow circle indicates the loss modulus. The graph shows the change in shear modulus (Pa) over time when the first and second parts of the adhesive using compound A obtained when the reaction time between genipin and polyamine was 30 minutes was set to 0 minutes. The solid circle indicates the storage modulus, and the hollow circle indicates the loss modulus. The graph shows the change in shear modulus (Pa) over time when the first and second parts of the adhesive using compound A obtained when the reaction time between genipin and polyamine was 60 minutes was set to 0 minutes. The solid circle indicates the storage modulus, and the hollow circle indicates the loss modulus. This shows the change in shear modulus (Pa) over time when the first and second parts of the adhesive are mixed at 0 minutes when the pH of the UBM (urinary bladder matrix) solution is 2.0. The solid circles represent the storage modulus and the hollow circles represent the loss modulus. This shows the change in shear modulus (Pa) over time when the first and second parts of the adhesive are mixed at 0 minutes when the pH of the UBM solution is 2.5. The solid circles represent the storage modulus and the hollow circles represent the loss modulus. This shows the change in shear modulus (Pa) over time when the first and second parts of the adhesive are mixed at 0 minutes when the pH of the UBM solution is 3.0. The solid circles represent the storage modulus and the hollow circles represent the loss modulus. This shows the change in shear modulus (Pa) over time when the first and second parts of the adhesive are mixed at 0 minutes when the pH of the UBM solution is 5.0. The solid circles represent the storage modulus and the hollow circles represent the loss modulus. This shows the change in shear modulus (Pa) over time when the first and second parts of the adhesive are mixed at 0 minutes when the pH of the UBM solution is 7.0. The solid circles represent the storage modulus and the hollow circles represent the loss modulus. This is the result of measuring the swelling degree of Hydrokel. This is the stress-strain relationship of the hydrogel. FIG. 1 shows cell viability (%). FIG. 13 is a graph showing adhesiveness to a collagen casing. 1 shows the results of adhesiveness evaluation using porcine aorta. 1 shows the results of histological evaluation of the porcine aorta after pressure testing. This shows the change over time in shear modulus (storage modulus: Pa) when the time when the first and second parts of the adhesive containing the second part containing UBM are mixed is set to 0 minutes. This shows the change over time in shear modulus (storage modulus: Pa) when the time when the first and second parts of the adhesive are mixed is set to 0 minutes, and the second part contains ECM (extracellular matrix) derived from the heart. This shows the change over time in shear modulus (storage modulus: Pa) when the time when the first and second parts of the adhesive containing the second part containing liver-derived ECM are mixed is set to 0 minutes. This shows the change over time in shear modulus (storage modulus: Pa) when the first and second parts of the adhesive, which includes a second part containing pancreatic-derived ECM, are mixed at 0 minutes. This shows the change over time in shear modulus (storage modulus: Pa) when the time when the first and second agents of the adhesive containing the second agent containing ECM derived from the small intestine are mixed is set to 0 minutes. This shows the change over time in shear modulus (storage modulus: Pa) when the time when the first and second parts of the adhesive are mixed is set to 0 minutes, and the second part of the adhesive contains a second part consisting only of PBS (phosphate-buffered saline). FIG. 13 is a diagram showing the adhesiveness of hydrogels prepared using second agents containing various ECMs to a collagen casing. FIG. 1 is a diagram showing the introduction rate of genipin (the introduction rate of the group represented by formula 1) relative to the amount (equivalent) of genipin charged. This shows the change over time in the shear modulus (storage modulus: Pa) when the first and second parts of the adhesive, which contains a first part with a 9% introduction rate of genipin, are mixed at 0 minutes. This shows the change over time in the shear modulus (storage modulus: Pa) when the first and second parts of the adhesive, which contains a first part with an introduction rate of 18% genipin, are mixed at 0 minutes. This shows the change over time in shear modulus (storage modulus: Pa) when the first and second parts of the adhesive, which contains a first part with a genipin introduction rate of 31%, are mixed at 0 minutes. This shows the change over time in the shear modulus (storage modulus: Pa) when the first and second parts of the adhesive, which contains a first part with a genipin introduction rate of 33%, are mixed at 0 minutes.

The present invention will be described in detail below.
The following description of the components may be based on a representative embodiment of the present invention, but the present invention is not limited to such an embodiment.
In this specification, a numerical range expressed using "to" means a range that includes the numerical values before and after "to" as the lower and upper limits.

In the description of groups (atomic groups) in this specification, when a notation does not indicate whether it is substituted or unsubstituted, it includes both unsubstituted and substituted groups, as long as it does not impair the effects of the present invention. For example, an "alkyl group" includes not only an alkyl group that does not have a substituent (unsubstituted alkyl group), but also an alkyl group that has a substituent (substituted alkyl group). This also applies to each compound.

[Two-component curing adhesive]
A two-component curing adhesive according to an embodiment of the present invention (hereinafter also simply referred to as "the adhesive") is a two-component curing adhesive comprising compound A having, within the molecule, at least one primary amino group and at least one group represented by formula 1 when * is the bonding position, a first component containing a basic compound, and a second component containing an acidic compound.

The mechanism by which the above two-component curing adhesive solves the problem of the present invention is not entirely clear, but the inventor speculates as follows. Note that the following speculation does not limit the technical scope of the present invention, and even if the problem is solved without relying on the following mechanism, the two-component curing adhesive etc. specified by the matters described in each claim are considered to be included in the scope of the present invention.

In developing a two-component curing adhesive that cures quickly, the present inventors have intensively investigated the crosslinking (curing) mechanism of genipin and primary amines.
In general, the crosslinking (curing) reaction mechanism of genipin is known to be (1) a nucleophilic attack on the C3 carbon atom of genipin by a primary amine, followed by ring-opening of the dihydropyran ring, followed by attack of the aldehyde group generated by the ring-opening by the secondary amine generated by the nucleophilic attack to form an N-containing heterocycle (dihydropyridine ring) (reaction scheme (1) in the formula below); (2) an SN2 nucleophilic substitution reaction in which an ester group is replaced by a secondary amide bond (reaction scheme (2) in the formula below); and (3) a dimerization reaction that occurs in the presence of oxygen radicals (it is believed that there are multiple forms of dimerization reaction).

The inventors came up with the idea that among these reactions, reaction scheme (1) and reaction scheme (2), which are easier to control even in an atmospheric environment and are assumed to contribute to the formation of the main crosslinking structure in the cured product, could be applied to a two-component curing adhesive that cures more quickly if the reactions are caused to occur in stages.

It was already known that the reaction rates of reaction scheme (1) and reaction scheme (2) may vary depending on various factors. However, the present inventors have recently discovered that the reaction of reaction scheme (1) is more likely to proceed in a solution containing a basic compound, for example, in an aqueous solution having a pH of more than 7.0 (preferably a pH of 11.0 or less), while the reaction of reaction scheme (2) is more likely to proceed in a solution containing an acidic compound, specifically, in an aqueous solution having a pH of less than 7.0 (preferably a pH of 2.0 or more), and have focused on this finding.

Thus, the inventors have completed a two-component curing adhesive in which a genipin derivative having a primary amino group is synthesized, the pH is adjusted with a basic compound (preferably to a pH greater than 7.0) to form a first component, which is then mixed with a second component containing an acidic compound to lower the pH (preferably to a pH less than 7.0), and the adhesive is cured by initiating the reaction according to reaction scheme (4) in the following formula.

Compound A contained in the first agent has a group represented by the above formula 1. The group represented by formula 1 is a group generated by the reaction of genipin with a primary amine according to reaction scheme (1). Surprisingly, the inventors have discovered that this group causes the reaction of reaction scheme (4) to proceed very quickly by lowering the pH of the reaction solution.

The following reaction formulas are schematic, and reactions derived from the dimerization mechanism of genipin may occur in parallel before, after, or during each reaction, but they are omitted. In the formulas, R ^A and R ^B each represent a monovalent group.

The inventors also investigated the opposite approach, that is, proceeding with the reaction scheme (2) in a low pH range, mixing with a pH adjuster, raising the pH, and proceeding with the reaction scheme (5) in a high pH range to promote crosslinking, but did not achieve the desired effect.

In other words, it is speculated that the adhesive achieves the desired effect by utilizing the relationship between the pH of the reaction solution and the crosslinking reaction rate of genipin, and by preparing in advance compound A having a group represented by formula 1 that facilitates the rapid reaction of reaction scheme (4), and using this as a crosslinking agent.

Note that the reaction scheme (4) in the above reaction formula shows a form in which a compound having a primary amino group, "H ₂ N-R ^B ," is bonded to compound A to form a crosslinked structure. This "H ₂ N-R ^B " may be compound A itself. In addition, as described later, "H ₂ N-R ^B " may be another compound contained in the first agent or the second agent (preferably the second agent). The components contained in the two-component curing adhesive according to the embodiment of the present invention will be described in detail below.

[First Agent]
The first agent contains compound A and a basic compound, and may further contain a solvent.

<Compound A>
Compound A is a compound having one or more primary amino groups and one or more groups represented by formula 1 in the molecule. (Note that in the formula, * indicates a bonding position.)

The content of compound A in the first agent is not particularly limited, but is generally preferably 1 to 99 mass% based on the total mass of the solids in the first agent, since this results in a two-component curing adhesive that has a more excellent effect of the present invention. The first agent may contain one type of compound A alone, or may contain two or more types. When the first agent contains two or more types of compound A, it is preferable that the total content is within the above numerical range.

The number of primary amino groups that compound A has is not particularly limited, but is preferably 2 or more, and more preferably 3 or more. There is no particular upper limit, but is preferably 50 or less, preferably 10 or less, more preferably 8 or less, and even more preferably 6 or less.

The number of groups represented by formula 1 in one molecule of compound A is not particularly limited, but is preferably 2 or more, and there is no particular upper limit, but is preferably 50 or less, more preferably 10 or less, more preferably 8 or less, even more preferably 6 or less, and particularly preferably 4 or less.

The content ratio of the molar content of the group represented by formula 1 to the molar content of the primary amino group in compound A (group represented by formula 1/amino group) is not particularly limited, but is generally preferably 0.01 to 2.0. From the viewpoint of obtaining a two-component curing adhesive having better effects of the present invention, 0.05 to 1.2 is preferable. In particular, when compound A is a compound represented by formula 1 described later, from the viewpoint of obtaining a two-component curing adhesive having better effects of the present invention, 0.4 to 1.0 is more preferable, and 0.5 to 0.9 is even more preferable.
On the other hand, when compound A is obtained by reacting gelatin with genipin as a polyamine described later, the content ratio is preferably 0.05 to 0.6.

Compound A has at least one primary amino group and at least one group represented by formula 1 in the molecule. As already explained, the group represented by formula 1 is typically a group formed by bonding genipin with a primary amine, and compound A is a genipin derivative.

As shown in the Examples (FIG. 22), the inventors' investigations have revealed that free genipin (shown as "UBM+Genipinn" in FIG. 22), which was previously thought to have low cytotoxicity, does not have sufficiently low cytotoxicity.
On the other hand, in the two-component curing adhesive of this embodiment, the component (compound A) that contributes to crosslinking is a genipin "derivative." In other words, genipin, which has cytotoxicity, is fixed to the skeletal structure described below, and has the excellent feature that the toxicity of genipin itself (free genipin) is unlikely to occur regardless of the progress of the crosslinking reaction.

Compound A is typically a compound having a skeleton structure, a primary amino group bonded to the skeleton structure, and a group represented by formula 1.
This skeleton structure is not particularly limited, and structures derived from various organic compounds can be used.
For example, the skeletal structure may be derived from polysaccharides, peptides, proteins, gelatin, ethylenediamine, polyethyleneimine, polyalkylene glycol, polylactic acid, polyglycolic acid, polycaprolactone, polyacrylic acid, or the like.

Among these, from the viewpoint of obtaining a two-component curing adhesive having a more excellent effect of the present invention, the backbone structure is preferably a structure derived from at least one selected from the group consisting of ethylenediamine, polyethyleneimine, polyalkylene glycol, polylactic acid, polyglycolic acid, polycaprolactone, and polyacrylic acid, more preferably a structure derived from at least one selected from the group consisting of polyalkylene glycol, polylactic acid, polyglycolic acid, polycaprolactone, and polyacrylic acid, and even more preferably a structure derived from polyalkylene glycol.

As compound A, a compound represented by formula 2 is preferred from the viewpoint of obtaining a two-component curing adhesive having a more excellent effect of the present invention.

In formula 2, s1 is an integer of 1 or more, and although there is no particular upper limit, it is preferably 8 or less, more preferably 6 or less, even more preferably 4 or less, and particularly preferably 2 or less.
In formula 2, t1 is an integer of 1 or more, preferably 2 or more, and although there is no upper limit, it is preferably 10 or less, more preferably 8 or less, even more preferably 6 or less, and particularly preferably 4 or less.
In formula 2, u1 is an integer of 0 or more, preferably 2 or less, and more preferably 1 or less.

M ¹ represents a group having a valence of s1+t1+u1. When M ¹ is a divalent group, examples of M ¹ include -C(O)-, -C(O)O-, -OC(O)-, -O-, -S-, -NR ²⁰ - (R ²⁰ represents a hydrogen atom or a monovalent organic group), an alkylene group (preferably having 1 to 10 carbon atoms), a cycloalkylene group (preferably having 3 to 10 carbon atoms), an alkenylene group (preferably having 2 to 10 carbon atoms), and combinations thereof.
Of these, from the viewpoint of obtaining a two-component curing adhesive having superior effects of the present invention, -C(O)-, -NH-, -O-, alkylene groups having 1 to 5 carbon atoms, and combinations of these are preferred.

Furthermore, when M ¹ is a trivalent or higher group, it is not particularly limited, and examples thereof include groups represented by (3a) to (3d).

In formula 3a, ^Q3 represents a trivalent group. ^T3 represents a single bond or a divalent group, and three ^T3 may be the same or different.
Examples of _Q3 include a tertiary amino group, a trivalent hydrocarbon group (preferably having 1 to 10 carbon atoms. The hydrocarbon group may be an aromatic hydrocarbon group or an aliphatic hydrocarbon group), or a trivalent heterocyclic group (preferably a 5- to 7-membered heterocyclic group), and the hydrocarbon group may contain a heteroatom (for example, -O-). Specific examples of _Q3 include a glycerin residue, a trimethylolpropane residue, a phloroglucinol residue, and a cyclohexanetriol residue.
The divalent group of ^T3 is not particularly limited, but may be the same as the divalent group of ^M1 already described, and among them, an alkylene group having 1 to 5 carbon atoms which may have a substituent is preferable.

In formula 3b, ^Q4 represents a tetravalent group. ^T4 represents a single bond or a divalent group, and the four ^T4s may be the same or different from each other.
Examples of ^Q4 include a tetravalent hydrocarbon group (preferably having 1 to 10 carbon atoms. The hydrocarbon group may be an aromatic hydrocarbon group or an aliphatic hydrocarbon group) and a tetravalent heterocyclic group (preferably a 5- to 7-membered heterocyclic group), and the hydrocarbon group may contain a heteroatom (for example, -O-). Specific examples of ^Q4 include a pentaerythritol residue and a ditrimethylolpropane residue.
The divalent group of ^T4 is not particularly limited, but may be the same as the divalent group of ^M1 already described, and among them, -O-, an alkylene group having 1 to 5 carbon atoms, or a combination thereof is preferable.

In formula 3c, ^Q5 represents a pentavalent group. ^T5 represents a single bond or a divalent group, and the five ^T5s may be the same or different from one another.
^Q5 may be a pentavalent hydrocarbon group (preferably having 2 to 10 carbon atoms. The hydrocarbon group may be an aromatic hydrocarbon group or an aliphatic hydrocarbon group) or a pentavalent heterocyclic group (preferably a 5- to 7-membered heterocyclic group), and the hydrocarbon group may contain a heteroatom (for example, -O-). Specific examples of ^Q5 include an arabinitol residue, a phloroglucidol residue, and a cyclohexanepentanol residue.
The divalent group of ^T5 is not particularly limited, but may be the same as the divalent group of ^M1 already described, and the preferred embodiments are also the same.

In formula 3d, ^Q6 represents a hexavalent group. ^T6 represents a single bond or a divalent group, and the six ^T6 may be the same or different from each other.
_Q6 may be a hexavalent hydrocarbon group (preferably having 2 to 10 carbon atoms. The hydrocarbon group may be an aromatic or aliphatic hydrocarbon group) or a hexavalent heterocyclic group (preferably a 6- to 7-membered heterocyclic group), and the hydrocarbon group may contain a heteroatom (for example, -O-). Specific examples of ^Q6 include a mannitol residue, a sorbitol residue, a dipentaerythritol residue, a hexahydroxybenzene residue, and a hexahydroxycyclohexane residue.
The divalent group of ^T6 is not particularly limited, but may be the same as the divalent group of ^M1 already described, and the preferred embodiments are also the same.

When M ¹ is a group having a valence of 7 or more, a group obtained by combining the groups represented by formulae 3a to 3d can be used.

In formula 2, L ¹ , L ² , and L ³ each independently represent a single bond or a divalent group. The divalent group is not particularly limited, and examples of the divalent group include the same groups as those already described as the divalent group of M ¹ .
Furthermore, L ¹ , L ² and L ³ preferably contain an alkyleneoxy group (oxyalkylene group) having a carbon number of 1 to 10. The group containing an alkyleneoxy group also includes a polyoxyalkylene group.
When L ¹ , L ² , and L ³ each contain an alkyleneoxy group (oxyalkylene group) having 1 to 10 carbon atoms, the cured product of the adhesive has superior hydrophilicity, and as a result, superior tissue adhesion and/or biocompatibility.

Among these, from the viewpoint of obtaining a two-component curing adhesive having more excellent effects of the present invention, the divalent group for L ¹ , L ² , and L ³ is preferably a group represented by the following formula (4).

In formula 4, R ¹ each independently represents an alkylene group having 1 to 10 carbon atoms (more preferably 2 to 6 carbon atoms), which may have a substituent, and is more preferably -CH ₂ CH ₂ -. L ⁴ represents a single bond or a divalent group, and examples of the divalent group of L ⁴ include the same groups as those described as the divalent group of M ¹ .
Furthermore, p1 represents a number of 2 or more, and is not particularly limited, but is more preferably 50 or more, even more preferably 100 or more, and is preferably 10,000 or less, more preferably 5,000 or less, even more preferably 2,000 or less, and particularly preferably 1,000 or less.
Additionally, * represents the bonding position to ^M1 , and ** represents the bonding position to the other group.
Although L ¹ , L ² and L ³ may be the same or different, it is preferable that they are all the same.

In addition, in formula 2, ^Y1 is a hydrogen atom or a monovalent group that does not have a primary amino group. The monovalent group is not particularly limited, and examples thereof include a hydrocarbon group that may have a hetero atom, a halogen atom, a carboxy group, a hydroxy group, and an alkoxy group. In addition, ^Y1 may be a group represented by the following formula. In the formula, * represents a bonding position.

In addition, in formula 2, X ¹ is a group represented by formula 1.

From the viewpoint of obtaining a two-component curing adhesive having a more excellent effect of the present invention, compound A is preferably a compound represented by the following formula 5.

In formula 5, X ¹ , L ¹ , and L ² are each defined as the same as each symbol in formula 2, and the preferred embodiments are also the same. ^{M 5} is a tetravalent group, and specific examples and preferred embodiments are the same as those of the tetravalent group of M ¹ in formula 2. In addition, s 5 is an integer of 1 to 3.

The molecular weight of compound A is not particularly limited, but typically is preferably 100 to 300,000, more preferably 1000 to 200,000, and even more preferably 2000 to 100,000.

(Method of synthesizing compound A)
Compound A can typically be obtained by reacting genipin with a polyamine (which in this specification means a compound containing two or more primary amino groups) in a basic solution.

As polyamines, any polyamine having multiple primary amino groups, such as chitosan, albumin, gelatin, ethylenediamine, and polyethyleneimine, can be used. In addition, polyalkylene glycols, polylactic acids, polyglycolic acids, polycaprolactones, and polyacrylic acids into which primary amino groups have been introduced can also be used.

The polyamine is preferably a compound represented by the following formula 6, since the resulting two-component curing adhesive containing compound A has a more excellent effect of the present invention.

In formula 6, s6 represents an integer of 2 or more, and although there is no particular upper limit, it is preferably 9 or less, more preferably 7 or less, even more preferably 5 or less, and particularly preferably 3 or less.
In formula 2, u6 represents an integer of 0 or more, preferably 2 or less, and more preferably 1 or less.

In formula 6, M ¹ , L ¹ , L ³ and Y ¹ have the same meanings as the respective symbols in formula 2, and the preferred embodiments are also the same.
Among them, Y ¹ is preferably a hydrogen atom, a hydroxyl group, or the following groups: In the following formulae, * indicates a bonding position.

Specific examples of polyamines include polysaccharides having amino sugars as structural units, such as chitosan; and proteins, such as elastin, albumin, and collagen. In addition, gelatin, which has a primary amino group derived from lysine, can also be used as a polyamine.

Other examples of polyamines include ethylenediamine and polyethyleneimine. In this case, the molecular weight of the polyethyleneimine is not particularly limited, but is preferably 300 to 100,000 in order to obtain a better effect of the present invention.

As another example of the polyamine, a derivative in which a plurality of primary amino groups are introduced into at least one polymer selected from the group consisting of polyalkylene glycol, polylactic acid, polyglycolic acid, polycaprolactone, and polyacrylic acid can also be used.
Since the polyamine has superior hydrophilicity and superior biocompatibility compared to the above polymers, when the above polyamine is used, the cured product of the two-component curing adhesive has superior biocompatibility.

There are no particular limitations on the molecular weight of the polyamine, but a molecular weight of 300 or more is preferred in that the resulting cured product will have better mechanical properties, and a molecular weight of 200,000 or less is preferred in that the first agent will have better fluidity.

<Basic Compound>
The first agent contains a basic compound. In this specification, a basic compound means a compound that, when 0.05 mol of the compound is dissolved in 1 kg of ultrapure water at 25° C. in an inert gas atmosphere not containing carbon dioxide gas, causes the pH of the aqueous solution to exceed 7.0 (rounded up to one decimal place).

The basic compound is not particularly limited, but examples thereof include sodium hydroxide, potassium hydroxide, sodium hydrogen carbonate, sodium carbonate, disodium hydrogen phosphate, and sodium borate.
The form in which the first agent contains a basic compound is not particularly limited, and for example, a pH adjuster (sodium hydroxide, etc.) or a buffer (disodium hydrogen phosphate, etc.) used in the synthesis process of compound A may ultimately remain in the first agent.

More specifically, compound A is preferably obtained by reacting genipin with a polyamine in an alkaline aqueous solution, and in this case, the solution contains a basic compound as a pH adjuster or buffer. The first agent may be in a form that contains a basic compound due to the basic compound used in the synthesis process of compound A remaining.

The content of the basic compound in the first agent is not particularly limited, but is generally preferably 0.0001 to 99% by mass based on the total mass of the solid content of the first agent, since this results in a two-component curing adhesive that has a better effect of the present invention. The first agent may contain one type of basic compound alone, or may contain two or more types. When the first agent contains two or more types of basic compounds, it is preferable that the total content is within the above numerical range.

<Solvent>
The first agent may contain a solvent. The solvent is not particularly limited, but is preferably water, a water-soluble organic solvent such as alcohol, or a mixture thereof, and more preferably water.
The content of the solvent in the first agent is not particularly limited, but from the viewpoints of handleability and curability, the solid content of the first agent is preferably adjusted to 0.01 to 99 mass%, more preferably 0.1 to 40 mass%, and still more preferably 1 to 30 mass%.
When the first agent contains a solvent, the pH of the first agent is not particularly limited, but it is preferably greater than pH 7.0 and less than pH 11.

The first agent may contain other ingredients within the scope of the effects of the present invention. The other ingredients are the same as the "other ingredients" that may be contained in the second agent, and will be described later.

[Second Agent]
The second agent includes an acidic compound. In this specification, the acidic compound means a compound that, when 0.05 mol of the compound is dissolved in 1 kg of ultrapure water at 25° C. in an inert gas atmosphere not containing carbon dioxide gas, gives an aqueous solution with a pH of less than 7.0 (rounded up to one decimal place).

The content of the acidic compound in the second agent is not particularly limited, but in relation to the first agent, it is preferable that the pH of the mixed solution be adjusted to 8.0 or less when the two agents (including the solvent) are mixed at the time of use.
The pH of the mixed solution is affected by the type and amount of the basic compound contained in the first agent, and by the type and amount of the solvent if the first agent contains a solvent, as well as by the temperature of the mixed solution during use.
Therefore, the content of the acidic compound may be appropriately selected in accordance with the above conditions. Such adjustment of pH is easy for those skilled in the art.

In order to make the two-component curing adhesive cure more quickly, it is more preferable that the pH of the mixture of the two components is adjusted to 7.0 or less, and more preferably to less than 7.0.
On the other hand, from the viewpoint of ensuring a sufficient time from preparation of the mixed liquid to curing (sufficient pot life), it is preferable to adjust the pH of the mixed liquid to 2.0 or more, more preferably to adjust the pH to more than 2.0, and even more preferably to adjust the pH to 5.0 or more.

The pH of the second agent is not particularly limited, but from the viewpoint of ease of adjusting the pH of the mixed solution, it is preferably less than 7.0, more preferably 5.0 or less, more preferably less than 5.0, even more preferably 3.0 or less, preferably 2.0 or more, more preferably more than 2.0, even more preferably 2.5 or more.

The acidic compound is not particularly limited, but examples thereof include hydrochloric acid, acetic acid, boric acid, citric acid, and citrate salts.
The content of the acidic compound in the second agent is not particularly limited, but is generally preferably 0.001 to 99 mass% based on the total mass of the solid content of the second agent, in order to obtain a two-agent curing adhesive having a better effect of the present invention. The first agent may contain one type of acidic compound alone, or may contain two or more types. When the first agent contains two or more types of acidic compounds, it is preferable that the total content is within the above numerical range.

<Solvent>
The second agent may contain a solvent. The solvent is not particularly limited, but is preferably water, a water-soluble organic solvent such as alcohol, or a mixture thereof, and more preferably water.
The content of the solvent in the second agent is not particularly limited, but from the viewpoints of handleability and curability, the solid content of the second agent is preferably adjusted to 0.01 to 99 mass%, and more preferably adjusted to 0.01 to 10 mass%.

Examples of water-soluble organic solvents include alcohols with 5 or fewer carbon atoms, such as methanol, ethanol, and isopropyl alcohol, and aprotic polar solvents, such as dimethyl sulfoxide.

<Other ingredients>
The second agent may contain ingredients other than those mentioned above.
An example of the above component is the polyamine described above as a compound that can be used in the synthesis of compound A. When the second part contains a polyamine, a two-part curing adhesive can be obtained that cures faster and/or can form a cured product with better adhesive properties.

The other component is preferably an extracellular matrix.
As used herein, the term "extracellular matrix" (ECM) is synonymous with "extracellular matrix" and refers to a substance present between somatic cells, whether epithelial cells or non-epithelial cells.
Extracellular matrix is a biological material generally produced by cells. It is involved in the formation of the internal environment necessary for the survival of somatic cells as well as the support of tissues. Extracellular matrix is generally produced by connective tissue cells, but some is also secreted by cells themselves that possess a basement membrane, such as epithelial cells and endothelial cells.

The extracellular matrix is broadly divided into fibrous components and the matrix that fills the gaps between them. The fibrous components include collagen fibers and elastic fibers. The basic component of the matrix is glycosaminoglycan (acidic mucopolysaccharide), most of which binds to non-collagenous proteins to form polymers of proteoglycan (acidic mucopolysaccharide-protein complex). In addition, the matrix also contains glycoproteins such as laminin in the basement membrane, microfibrils around elastic fibers, fibers, and fibronectin on the cell surface. Representative examples of extracellular matrices include collagen I, collagen III, collagen V, elastin, vitronectin, fibronectin, laminin, thrombospondin, and proteoglycans (e.g., decorin, biglycan, fibromodulin, lumican, hyaluronic acid, aggrecan, etc.).

When the second part contains an extracellular matrix, the cured product of the two-part curing adhesive has better tissue regeneration ability, better tissue adhesion, and cures faster.

The extracellular matrix may be purchased or may be prepared by a known method. The method for preparing the extracellular matrix is not particularly limited, but for example, a method can be used in which an organ derived from a living body is washed, then a process for removing nucleic acids as necessary, and then digested with acid proteases such as trypsin and pepsin in an acidic solution, and the solvent is removed from the resulting digestive solution by a method such as freeze-drying. Also, the matrix secreted by cultured cells can be used.
When the second agent contains an extracellular matrix, the acidic compound contained in the second agent may be one that is contained (transferred) in the second agent as a result of the preparation process of the extracellular matrix, due to its preparation method.

The type of organ derived from a living body used to prepare the extracellular matrix is not particularly limited, but examples include the urinary bladder, heart, liver, pancreas, and small intestine of a mammal. From the viewpoint of providing the resulting two-component curing adhesive with superior adhesive properties (compression resistance: FIG. 32), at least one selected from the group consisting of the urinary bladder, heart, liver, and small intestine is preferred, and at least one selected from the group consisting of the urinary bladder and small intestine is more preferred. In addition, examples of cultured cells include fibroblasts derived from human fetuses.

When the second agent contains an extracellular matrix, the content of the extracellular matrix in the second agent is not particularly limited, but is generally, when the total mass of the solids in the second agent is taken as 100 mass%, preferably, but not particularly limited to, 0.1 to 99.99 mass%, and may be appropriately adjusted depending on the content of other components, particularly acidic compounds, contained in the second agent.

The second agent may contain various other drugs in addition to those mentioned above.
When the second part contains these drugs, the cured adhesive can be used as a local delivery vehicle or a sustained release delivery vehicle for the drugs.
Drugs include, but are not limited to, anticancer drugs, anti-inflammatory drugs, antithrombotic drugs, antibiotics, and biological agents, as well as growth factors such as fibroblast growth factor, vascular endothelial growth factor, and hepatocyte growth factor.
Furthermore, when a virus and/or a cancer antigen protein is contained as a vaccine, the cured adhesive can be used as a vaccine carrier.

[Configuration of two-component curing adhesive]
This adhesive has a first agent and a second agent, and by mixing them, the curing reaction proceeds and the adhesive cures quickly. Moreover, the pot life can be freely controlled by adjusting the relative relationship between the content of the basic compound in the first agent and the content of the acidic compound in the second agent, that is, by adjusting the degree to which the pH of the mixture is lowered (details are shown in the examples). Therefore, when the purpose is to repair a damaged area in a living body, the adhesive can be directly injected into the damaged area with a syringe or the like after mixing the two agents, and the adhesive can be quickly cured at the damaged area by adjusting the degree to which the pH of the mixture is lowered, taking into consideration the working time in advance.

The mass ratio (percentage) of the content of the first agent to the total solid content of the two-agent curing adhesive is not particularly limited, but is preferably 0.01 mass% or more, more preferably 70 mass% or more, even more preferably 80 mass% or more, particularly preferably 90 mass% or more, most preferably 95 mass% or more, and preferably 99.99 mass% or less.

On the other hand, the content of the second agent relative to the total solid content of the two-agent curing adhesive is not particularly limited, but in general, it is preferably 0.01% by mass or more, more preferably 30% by mass or less, more preferably 20% by mass or less, even more preferably 10% by mass or less, and particularly preferably 5% by mass or less.

Since this adhesive has a first agent and a second agent, these may be filled in advance into a syringe to make a prefilled syringe. In that case, as shown in the examples below, the mechanical properties, crosslinking time, pot life, etc. can be freely controlled, so it can be used for healing muscle damage, preventing pancreatic leakage, preventing thinning after myocardial infarction, preventing adhesions, treating spinal cord injuries, treating bedsores, and wound healing in the gastrointestinal mucosa.

There are no particular limitations on the method for curing this adhesive, but for example, it is preferable to leave it at a temperature of 20 to 40°C for about 10 minutes to 2 hours, and more preferably for 30 to 60 minutes.

[Cured product]
The cured product of the adhesive has a three-dimensional network structure due to a crosslinked structure formed by compound A and, if necessary, an extracellular matrix, etc. From the viewpoint of biocompatibility and tissue regeneration ability, the cured product is preferably a hydrogel containing water. The water may be derived from the first agent and/or the second agent, or may be present around the adherend.

Hydrogel is a gel that contains water and has no fluidity. The water content in the hydrogel is not particularly limited, but is preferably 80 to 99% by weight when the total mass of the hydrogel is 100% by mass.

[Uses of two-component curing adhesives]
The adhesive can be rapidly cured using the genipin derivative, and when the second agent contains ECM, it can form a cured product (hydrogel) with higher biocompatibility, higher tissue adhesiveness, and higher tissue regeneration ability, and can be used as a medical tissue adhesive.

In addition, when the first or second agent contains a drug, the hardened material can be fixed to the lesion and used to release the drug. In particular, when the second agent contains ECM, it has higher biocompatibility and can be used as a scaffolding material for cells during cell transplantation in the field of regenerative medicine.

The present invention will be described below with reference to examples, but the present invention is not limited to these.

<Preparation of first agent containing compound A>
A 4-branched polyethylene glycol having an amino group at its terminal (manufactured by NOF AMERICA CORPORATION, product name "SUNBRIGHT PTE-200PA", molecular weight 20,000) was dispersed in a phosphate buffered saline (PBS, pH = 7.4). Hereinafter, this aqueous dispersion is referred to as the "polyamine liquid."

The above-mentioned "SUNBRIGHT PTE-200PA" corresponds to a polyamine represented by formula 6, and contains as a main component a compound (molecular weight 20,000) in which, in formula 6, M ¹ is a pentaerythritol residue, L ¹ is a group represented by formula 4, R ¹ in formula 4 is -CH ₂ CH ₂ -, L ⁴ is -CH ₂ CH ₂ CH ₂ -, u6 is 0, and s6 is 4.

When the amount of amino groups (n) in the 4-branched polyethylene glycol is taken as 100 mol %, 50-125 mol % of genipin was added to the polyamine liquid and stirred. Compound A was synthesized in the liquid by stirring at 20-30°C for 3-60 minutes, and a compound corresponding to formula 5 was obtained under all conditions. After the reaction was completed, the dispersion was flash frozen and the sample was stored. This corresponds to the first agent (solvent-containing form) of this adhesive. The first agent contains disodium hydrogen phosphate (buffer) and sodium hydroxide (pH adjuster).

Furthermore, the above dispersion was freeze-dried to obtain a dry powder containing compound A (and a basic compound). This corresponds to the first agent of this adhesive in a form that does not contain a solvent. In each of the following tests, this was redissolved in ultrapure water as necessary and used as a liquid first agent.

<Measurement using an ultraviolet-visible spectrophotometer>
When compound A was synthesized according to the above method, the number of genipin introduced per molecule of polyamine was measured using an ultraviolet-visible spectrophotometer. Genipin (70 mol%) and polyamine solution (polyamine content 20 mass%, pH = 10.5) were mixed, and samples were taken at each time (up to 60 minutes), and the amount of genipin introduced (the amount of the group represented by formula 1 introduced) was calculated from the absorption spectrum. As a calibration curve, genipin was reacted with ethanolamine and the absorption at 290 nm was measured.

As a result, the absorbance at 290 nm increased significantly one minute after mixing the polyamine solution with genipin, and the absorbance increased with the increase in reaction time.
FIG. 1 is a graph showing the results of calculation of the average amount of genipin (group represented by formula 1) introduced into one molecule of polyamine (unit: piece) against the reaction time (minutes) on the horizontal axis.
According to FIG. 1, it was found that in a reaction time of 30 minutes, an average of 1.7 genipin molecules (groups represented by formula 1) were introduced per one molecule of polyamine (4-branched polyethylene glycol).

<Effect of pH during synthesis of compound A on genipin introduction rate>
The pH of a polyamine solution (20% by mass) was changed from 2 to 10, and genipin (100% by mole) was dissolved therein and reacted. The number of genipin molecules introduced was measured using an ultraviolet-visible spectrophotometer.
A calibration curve was prepared by reacting genipin with ethanolamine and measuring the absorbance at 290 nm.

FIG. 2 is a graph showing the results of calculating the average amount of genipin introduced per polyamine molecule (the group represented by formula 1) versus the pH of the polyamine solution.
According to FIG. 2, the amount of genipin introduced is small on the low pH side, i.e., the reaction rate is significantly reduced. It was found that genipin is more easily introduced when the pH exceeds 7.0, and is even more easily introduced at a pH of 9.0 or higher.

<Curing speed test>
The second part was prepared according to the following procedure, and reacted with the first part to examine the hardening speed (gelation speed).
First, a decellularized matrix derived from a porcine urinary bladder (Urinary Bladder Matrix, UBM) was prepared as a component of the second agent.

The UBM was prepared as follows. First, the epithelial tissue of the pig bladder was excised with a scalpel and washed with physiological saline. Then, it was washed with a treatment solution (0.1% by weight peracetic acid, 4% ethanol) for 2 hours, and then washed with physiological saline and ultrapure water. Then, it was treated with a DNase solution (0.1 mg/mL) for 24 hours to remove DNA. It was further washed with physiological saline and ultrapure water, and 10 mg of the obtained powder was treated with 1 mg of pepsin and 1 mL of 0.01 M hydrochloric acid for 48 hours. PBS was added to adjust the pH to 7 to inactivate the pepsin, and the UBM powder was obtained by lyophilization.

The UBM powder was redissolved by adding ultrapure water, and the pH was adjusted by adding 1M hydrochloric acid to obtain a UBM solution. This UBM solution (1% by mass of UBM powder, pH = 2.5, containing hydrochloric acid, corresponding to the second agent) and a compound A solution (20% by mass of the compound A obtained above, pH = 10.5, corresponding to the first agent) were mixed in equal amounts and stirred at 37°C, and the time until hardening was measured. The hardening (hydrogelation) time was defined as the time when the stirrer stopped rotating. As a result, it was revealed that the solution hardened 30 seconds after mixing.
This was significantly faster than conventional genipin-containing adhesives, which took several hours to a day to harden.

<Viscosity measurement>
The viscoelasticity of the cured product (hydrogel) was measured using a viscoelasticity measuring device (rheometer "Rheoplus", Anton Paar).
100 μL of the pre-gel solution (a mixture of the first and second agents) was placed on the stage of the rheometer and clamped with a jig having a diameter of 10 mm. The temperature of the stage was kept at 37° C. during the measurement.

The results are shown in Figures 3 to 6. Figure 3 shows the shear modulus (Pa) that changes over time, with the mixing of the first and second parts of the adhesive taken as 0 minutes, where the solid circles (filled circles) represent the storage modulus and the hollow circles (open circles) represent the loss modulus.
As described above, Figures 4 to 6 show the results for only the second agent (UBM solution) in Figure 4, the mixture of the second agent and genipin in Figure 5, and the mixture of the second agent, genipin, and polyamine in Figure 6.

Although UBM (Figure 4) gels by collagen self-assembly even when used alone, it has a small storage modulus (G'). On the other hand, in the pre-gel solution (Figure 3) consisting of UBM solution (1% by mass, pH = 2.5) and compound A solution (20% by mass, pH = 10.5), G' rose immediately after mixing the first and second agents, and a hardened product (hydrogel) with high strength was formed.

On the other hand, the combinations of UBM/genipin (Figure 5) and UBM/genipin/polyamine (Figure 6) caused gelation, but the reaction time was slow.
These results demonstrated that the adhesive hardened quickly after the two components were mixed, and could easily form a decellularized matrix hydrogel.

Next, multiple compound A were synthesized by varying the amount of genipin to be reacted with polyamine, and compound A solutions (20% by mass, pH 10.5) were prepared from each of them. These were then mixed with UBM solution (1% by mass, pH 2.5) to prepare pregel solutions. Viscoelasticity measurements were performed on these pregel solutions in the same manner as above.

The results are shown in Figures 7 to 10. Figure 7 shows the change in shear modulus (Pa) over time when the first and second parts of the adhesive used compound A, which was synthesized by adding 50 mol % of genipin to 100 mol % of the primary amino groups of the polyamine, were mixed at 0 minutes. The solid circle indicates the storage modulus, and the hollow circle indicates the loss modulus.
As described above, Figures 8 to 10 show the results when the genipin concentrations were 60 mol%, 70 mol%, and 80 mol%, respectively.

7 to 10, when the amount of genipin added was 60 mol% or more, the curing (gelation) became faster. On the other hand, when the amount of genipin added was 70 mol% or less, the time from mixing the two components to curing (hereinafter referred to as "pot life") became longer.
At any equivalent ratio, the curing time was sufficiently fast.

Next, multiple compounds A were synthesized by varying the reaction time between polyamine and genipin, and a compound A solution (20% by mass, pH 10.5) was prepared, which was then mixed with a UBM solution (1% by mass, pH 2.5) to prepare a pregel solution. The viscoelasticity of this pregel solution was measured in the same manner as above.

The results are shown in Figures 11 to 14. Figure 11 shows the change in shear modulus (Pa) over time when the first and second parts of the adhesive using compound A obtained when the reaction time between genipin and polyamine was set to 3 minutes was set to 0 minutes. The solid circle indicates the storage modulus, and the hollow circle indicates the loss modulus.
As in the above, FIGS. 12 to 14 show the results when the reaction times were 10 minutes, 30 minutes, and 60 minutes, respectively.

Figures 11 to 14 show that curing is faster when genipin and polyamine are reacted for 10 minutes or more.

Next, the content of the acidic compound in the UBM solution (second agent) was adjusted to change the pH, and the solution was mixed with Compound A solution (20% by mass, pH 10.5) to prepare a pre-gel solution. Viscoelasticity measurements were performed on this pre-gel solution in the same manner as above.

The results are shown in Figures 15 to 19. Figure 15 shows the change in shear modulus (Pa) over time when the first and second parts of the adhesive were mixed at 0 minutes when the pH of the UBM solution was 2.0. The solid circles represent the storage modulus and the hollow circles represent the loss modulus.
As in the above, FIGS. 16 to 19 show the results when the pH of the UBM solution was 2.5, 3.0, 5.0, and 7.0, respectively.

Figures 15 to 19 show that when the pH of the UBM solution is less than 7.0, the hardening rate is faster, at 5.0 or less the hardening rate is even faster, at less than 5.0 the hardening rate is particularly faster, and at 3.0 or less the hardening rate is the largest. It was also found that when the pH of the UBM solution exceeds 2.0, a more sufficient pot life is obtained, and at 2.5 or more an even more sufficient pot life is obtained.

<Measurement of swelling degree>
Equal amounts of UBM solution (1% by mass, pH=2.5) and Compound A solution (20% by mass, pH=10.5) were mixed and gelled at 37° C. for 60 minutes. The resulting gel was cut into disks with a diameter of 5 mm, immersed in PBS, and allowed to stand at 37° C. to swell. The gel was collected at predetermined intervals, and the swollen weight (Ws) was measured. The gel was then freeze-dried to determine the dry weight (Wd). The degree of swelling was calculated from the formula: (Ws-Wd)/Wd.
As a result, it was found that the swelling degree of this hydrogel was about 17, and equilibrium swelling was reached in 1 hour (FIG. 20 shows the results).

<Mechanical strength measurement by tensile test>
Equal amounts of UBM solution (1% by weight, pH=2.5) and compound A solutions (5, 10, and 20% by weight, pH=10.5) were mixed, poured into a silicon mold of ISO 37-2 size, and cured (gelled) at 37° C. for 60 minutes.
Next, a tensile test was carried out on the prepared cured product (gel) using a texture analyzer.

21 shows the stress-strain relationship of the cured product. In the figure, 10%, 5%, and 2.5% respectively represent the contents of compound A in the first agent used to prepare the pre-gel solution.
From Fig. 21, it was found that the mechanical strength of the cured product (hydrogel) can be controlled by the content of compound A. From the above results, it was found that from the viewpoint of increasing the breaking strength of the cured product, the content of compound A in the mixed solution is preferably 75 mass% or more, and more preferably 85 mass% or more. In addition, it was found that from the viewpoint of increasing the maximum elongation of the cured product, the content of compound A is preferably 90 mass% or less, and more preferably 80 mass% or less.

<Cytotoxicity test of cured material (hydrogel)>
Cytotoxicity was evaluated by immersing the cured product (hydrogel) in a culture medium and adding the supernatant to the cells to be evaluated.
Equal amounts of UBM solution (1% by mass, pH = 2.5) and compound A solution (5, 10, 20% by mass, pH = 10.5) were mixed and cured (gelled) at 37°C for 60 minutes.

10 mL of RPMI (Roswell Park Memorial Institute medium) 1640 medium (10% fetal bovine serum, 1% penicillin-streptomycin) was added per 1 g of the cured product (hydrogel), and the mixture was allowed to stand at 37° C. for 24 hours, and the supernatant was collected.
The toxicity of the cured product (hydrogel) was evaluated using mouse fibroblast cells (L929 cells) as the evaluation cells.

L929 cells were cultured in an incubator at 37°C and 5% _CO2 using RPMI1640 medium (10% fetal bovine serum, 1% penicillin-streptomycin). 1 x ¹⁰⁴ L929 cells were seeded in a 96-well plate and pre-cultured for 24 hours. The collected supernatant or untreated medium alone as a control was added to each well and cultured for another 24 hours. After completion of the culture, the cell number was quantified using a cell counting kit (WST-8, DOJINDO).

Fig. 22 is a graph showing cell viability (%). From the results in Fig. 22, the hydrogel (shown as "UBM+GeniPEG" in Fig. 22) which is a cured product of UBM-compound A showed high cell viability.
In addition, a hydrogel not containing UBM (shown as "GeniPEG" in FIG. 22, which is similar to "UBM+GeniPEG" except that the second agent does not contain UBM) also showed high cell viability.

On the other hand, in the sample containing underivatized genipin and UBM (labeled "UBM + Genipin"), the cell viability was significantly lower.
This was thought to be due to the effect of genipin, as the "UBM" sample, which did not contain genipin, showed high cell viability.

Genipin has been considered to be a crosslinking agent with low cytotoxicity, but as mentioned above, according to the present inventor's study, the cell viability rate was not sufficiently low. However, in the compound A, which is the crosslinking agent contained in the first agent of the two-agent curing adhesive of the present invention, genipin is derivatized, and genipin is difficult to dissolve in the cured material. Therefore, it is presumed that the crosslinking function of genipin is maintained while the cytotoxicity is significantly reduced.
In other words, it was revealed that the hydrogel, which is the cured product of this adhesive, exhibits high cytocompatibility.

<Adhesion to collagen casing>
In accordance with ASTM-F2392-04R, adhesiveness evaluation was performed using a collagen casing as a model tissue for evaluating tissue adhesiveness.
A pinhole with a diameter of 3 mm was made in a collagen casing with a diameter of 35 mm, and 200 μL of the pregel solution was added.
The pre-gel solution was prepared by mixing 100 μL of the UBM solution (1% by mass, pH=2.5) and 100 μL of the compound A solution (20% by mass, pH=10.5).

This was left to stand at 37°C for 60 minutes and the compressive strength was measured. Figure 23 shows the results. As shown in Figure 23, the cured product of this adhesive (labeled "UBM + GeniPEG" in Figure 23) showed a high compressive strength of 50 mmHg. On the other hand, UBM alone was below the detection limit (0.75 mmHg or less), and "UBM + Genipin" showed a low value of 5.5 mmHg. These results make it clear that the cured product of this adhesive has high tissue adhesion.

<Adhesion to porcine aorta>
Adhesion evaluation was carried out using porcine aorta in accordance with ASTM-F2392-04R.
A pinhole with a diameter of 3 mm was created in a porcine aorta cut to a diameter of 30 mm, and 200 μL of pregel solution was added to the outside of the aorta. This was left to stand at 37° C. for 60 minutes, and the pressure resistance was measured. Figure 24 shows the results.
The pre-gel solution was prepared by mixing 100 μL of the UBM solution (1% by mass, pH=2.5) and 100 μL of the compound A solution (20% by mass, pH=10.5).

As shown in Figure 24, the cured product of this adhesive (shown as "UBM + GeniPEG" in Figure 24) exhibited a high pressure resistance of 100 mmHg. On the other hand, UBM alone was below the detection limit (0.75 mmHg or less), and "UBM + Genipin" had a low value of 3.3 mmHg. These results demonstrate that the cured product of this adhesive has high tissue adhesion even to the aorta.

Figure 25 shows the results of histological evaluation of the porcine aorta after the pressure test. Figure 25 reveals that "UBM + Genipin" did not remain on the tissue surface, whereas "UBM + GeniPEG" strongly adhered to the tissue surface and remained.

<Measurement of viscoelasticity of hydrogels prepared using second agents containing various ECMs>
A second agent containing a decellularized matrix (ECM) other than UBM was prepared, and the viscoelasticity of the cured product (hydrogel) was measured.

First, a portion of the pig heart, liver, pancreas, and small intestine was removed using a scalpel and washed with saline. After that, it was washed with a treatment solution (0.1% by weight peracetic acid, 4% ethanol) for 2 hours, and then washed with saline and ultrapure water. It was then washed with a 1% Triton/0.1% SDS solution for 24 hours, and then washed with ultrapure water. It was then treated with a DNase solution (0.5 mg/mL) for 24 hours to remove DNA. It was further washed with saline and ultrapure water, treated with pepsin (1 mg/mL) for 48 hours, and freeze-dried to obtain various matrix powders.

Using the above powder, 100 μL of pregel solution was prepared. The pregel solution was prepared by mixing 100 μL of ECM powder solution (ECM 1% by mass, pH = 2.5, second agent) prepared from UBM, heart, liver, pancreas, and small intestine, respectively, with 100 μL of compound A solution (20% by mass, pH = 10.5, first agent).

100 μL of the pre-gel solution was placed on the stage of the viscoelasticity measuring device and clamped with a jig having a diameter of 10 mm. The temperature of the stage was kept at 37° C. during the measurement.
The results are shown in Figures 26 to 31. Figure 26 shows the change over time in shear modulus (storage modulus: Pa) when the time when the first and second agents of the adhesive containing the second agent containing ECM derived from the bladder (UBM) is set to 0 minutes. As above, Figures 27 to 30 show the results for the adhesive containing the second agent containing ECM derived from the heart, liver, pancreas, and small intestine, respectively. Also, Figure 31 shows the results for the adhesive containing no ECM and a second agent consisting only of PBS (pH = 2.5).

26 to 31 show that all pre-gel solutions hardened (gelled) within 30 minutes. In particular, it was confirmed that the adhesive containing the second agent containing UBM or ECM derived from the small intestine hardened more quickly.
A comparison of Figures 26 to 30 with Figure 31 confirms that when the second agent contains ECM, curing occurs more rapidly.

<Adhesion of hydrogels prepared using second agents containing various ECMs to collagen casing>
In accordance with ASTM-F2392-04R, adhesiveness evaluation was performed using a collagen casing as a model tissue for evaluating tissue adhesiveness.
A pinhole with a diameter of 3 mm was made in a collagen casing with a diameter of 35 mm, and 200 μL of pregel solution was added. This was left to stand at 37° C. for 60 minutes, and the pressure resistance was measured. Figure 32 shows the results.
The pre-gel solution was prepared by mixing 100 μL of ECM powder solution (ECM: 1% by mass, pH=2.5, second agent) prepared from UBM, heart, liver, pancreas, and small intestine, respectively, with 100 μL of compound A solution (20% by mass, pH=10.5).

32, it was confirmed that each cured product (hydrogel) had a high pressure resistance strength of 40 mmHg or more. In particular, the cured product of the two-component curing adhesive containing the UBM and the second component containing ECM derived from the small intestine had a higher tissue adhesion strength.
In FIG. 32, "Heart" indicates ECM derived from the heart, "Liver" indicates ECM derived from the liver, and "Pancreas" indicates ECM derived from the pancreas.

<Measurement using an ultraviolet-visible spectrophotometer>
Compound A was synthesized in the same manner as above, except that a polyamine solution containing gelatin was used instead of the 4-branched polyethylene glycol.
At that time, in order to measure the number of genipin (groups represented by formula 1) introduced per molecule of polyamine (gelatin), measurement was performed using an ultraviolet-visible spectrophotometer.
Genipin was added to a gelatin solution (content 20% by mass, pH = 10.5, PBS) so that the amount of genipin was 10 to 40 mol % relative to the primary amino groups of gelatin, and mixed at 50°C. After 2 minutes, a sample was taken and the amount of genipin introduced (the amount of the group represented by formula 1 introduced) was calculated from the absorption spectrum. For the calibration curve, genipin was reacted with ethanolamine and the absorption at 290 nm was measured.

As a result, when gelatin solution was mixed with genipin, the absorption at 290 nm increased significantly, and the absorption increased as the amount of genipin added increased. Figure 33 shows the results of calculating the amount of genipin (group represented by formula 1) introduced (unit: %) to the amino groups in gelatin, with the horizontal axis representing reaction time (minutes) and the vertical axis representing the amount of genipin introduced (unit: %) to the amino groups in gelatin. It was found that the introduction rate increased as the amount of genipin added (equivalent to the amino groups) increased, and was 9% to 33%.

<Rheology>
Compound A was synthesized by varying the amount of genipin reacted with gelatin, and a compound A solution (20% by mass, pH 10.5, first agent) was prepared from each of them, which was then mixed with a UBM solution (1% by mass, pH 2.5, second agent) to prepare a pregel solution. Viscoelasticity of this pregel solution was measured in the same manner as above.

Figures 34 to 37 show the change over time in the shear storage modulus (Pa) of this adhesive, with the first agent containing compound A in which 9 to 33% genipin has been introduced relative to 100 mol % of the primary amino groups of gelatin, when the time when the first agent and the second agent are mixed is set to 0 minutes.

34 to 37, when the introduction rate of genipin was 18 mol% or more, the curing (gelation) became faster. On the other hand, when the introduction rate of genipin was less than 18 mol%, the pot life became longer after mixing of the two components.
The curing time was sufficiently fast at all equivalent ratios.

The hydrogel that is the cured product of the two-component curing adhesive of the present invention is a hydrogel that has high biocompatibility, tissue adhesiveness, and tissue regeneration ability, and is extremely useful as a tissue adhesive for medical applications.
In addition, by incorporating anticancer drugs into the hydrogel, it is expected that it can be used in cancer treatment. Furthermore, in the field of regenerative medicine, it can be used as a scaffold for cells during cell transplantation.

Claims (21)

a first agent including a compound A having, in the molecule, at least one primary amino group and at least one group represented by formula 1 when * is the bonding position, and a basic compound;
and a second agent containing an acidic compound.
At least one selected from the group consisting of the first agent and the second agent contains a solvent;
2. The two-component curing adhesive according to claim 1, wherein when the first and second components are mixed, the resulting mixture has a pH of 8.0 or less. The two-component curing adhesive according to claim 2, wherein the pH of the mixture is less than 7.0. The two-component curing adhesive according to claim 2 or 3, in which the pH of the mixture is 5.0 or higher. the second part comprises a solvent,
The two-component curing adhesive according to any one of claims 1 to 4, wherein the second part has a pH of less than 7.0. The two-component curing adhesive according to claim 5, wherein the pH of the second component is 2.0 to 5.0. The first agent includes a solvent,
The two-component curing adhesive according to any one of claims 1 to 6, wherein the pH of the first component is greater than 7.0. the compound A has a skeleton structure, the primary amino group bonded to the skeleton structure, and a group represented by formula 1;
The two-component curing adhesive according to any one of claims 1 to 7, wherein the skeletal structure is derived from at least one compound selected from the group consisting of polysaccharides, peptides, proteins, gelatin, ethylenediamine, polyethyleneimine, polyalkylene glycols, polylactic acids, polyglycolic acids, polycaprolactones, and polyacrylic acids. The two-component curing adhesive according to any one of claims 1 to 8, wherein compound A is a compound represented by the following formula 2:
(In formula 2, s1 is an integer of 1 or more, t1 is an integer of 1 or more, u1 is an integer of 0 or more, L ¹ , L ² , and L ³ are each independently a single bond or a divalent group, M ¹ is a s1+t1+u monovalent group, Y ¹ is a hydrogen atom or a monovalent group not having the primary amino group, and X ¹ is a group represented by formula 1.) 10. The two-component curing adhesive according to claim 9, wherein L ¹ , L ² , and L ³ each contain an alkyleneoxy group having 1 to 10 carbon atoms. The two-component curing adhesive according to any one of claims 1 to 10, wherein compound A is a compound represented by the following formula 5:
(In formula 5, S5 is an integer of 1 to 3, L ¹ and L ² are each independently a divalent group containing an alkyleneoxy group having 1 to 10 carbon atoms, X ¹ is a group represented by formula 1, and M ⁵ is a tetravalent group.) The two-component curing adhesive according to any one of claims 1 to 11, wherein the second component includes an extracellular matrix. The two-component curing adhesive according to any one of claims 2 to 7, in which the pH is 5.0 to 8.0 when the first and second components are mixed. The two-component curing adhesive according to any one of claims 1 to 13, wherein the ratio of the molar content of the group represented by formula 1 to the molar content of the primary amino group in compound A is 0.05 to 1.2. The two-component curing adhesive according to any one of claims 1 to 14, wherein compound A has 2 to 6 primary amino groups and 2 to 6 groups represented by formula 1. A curing agent for a two-component curing adhesive, comprising compound A having, in the molecule, at least one primary amino group and at least one group represented by formula 1 when * is the bonding position.
The hardener according to claim 16, further comprising a basic compound. the compound A has a skeleton structure, the primary amino group bonded to the skeleton structure, and a group represented by formula 1;
The curing agent according to claim 16 or 17, wherein the skeletal structure is derived from at least one compound selected from the group consisting of polysaccharides, peptides, proteins, gelatin, ethylenediamine, polyethyleneimine, polyalkylene glycols, polylactic acids, polyglycolic acids, polycaprolactones, and polyacrylic acids. A compound having a backbone structure, a primary amino group bonded to the backbone structure, and a group represented by formula 1:
the skeletal structure is derived from at least one compound selected from the group consisting of polysaccharide, gelatin, ethylenediamine, polyethyleneimine, polyalkylene glycol, polylactic acid, polyglycolic acid, polycaprolactone, and polyacrylic acid,
A compound represented by the following formula 2.
(In formula 2, s1 is an integer of 1 or more, t1 is an integer of 1 or more, u1 is an integer of 0 or more, L ¹ , L ² , and L ³ are each independently a single bond or a divalent group, M ¹ is a monovalent group of s1+t1+u, Y ¹ is a hydrogen atom or a monovalent group not having a primary amino group, and X ¹ is a group represented by formula 1 below when * is the bonding position.)
Said L ¹ , said L ² , and the L ³ The compound of claim 19, wherein comprises an alkyleneoxy group having 1 to 10 carbon atoms. The compound according to claim 19, represented by the following formula 5:
(In formula 5, S5 is an integer of 1 to 3, L ¹ , and L ² are each independently a divalent group containing an alkyleneoxy group having 1 to 10 carbon atoms; ¹ is a group represented by formula 1, M ⁵ is a tetravalent group.)